Bioconjugation is the process of coupling two biomolecules together in a covalent linkage. Common types of bioconjugation chemistry are amine coupling of lysine amino acid residues (typically through amine-reactive succinimidyl esters), sulfhydryl coupling of cysteine residues (via a sulfhydryl-reactive maleimide), and photochemically initiated free radical reactions, which have broader reactivity. The product of a bioconjugation reaction is a bioconjugate. The most common bioconjugations are coupling of a small molecule (such as biotin or a fluorescent dye) to a protein, or protein-protein conjugations, such as the coupling of an antibody to an enzyme. Other less common molecules used in bioconjugation are oligosaccharides, nucleic acids, synthetic polymers such as polyethylene glycol (a.k.a. PEG a.k.a. polyethylene oxide) and carbon nanotubes.
Bioconjugation methods rely heavily on chemoselective modification of native protein functional groups. Lysine and cysteine side chains are the most commonly functionalized amino acids. However, the high abundance of lysine on protein surfaces makes site-specific modification challenging. In contrast, cysteines are rare and are most often found in disulfide linked pairs in proteins in their natural environment. Labeling at this amino acid typically requires reduction of the target disulfide followed by reaction with a reagent like maleimide. Recently significant attention has been paid to the bioorthogonal modification of the aromatic amino acid side chains of tryptophan and tyrosine. Tyrosine modification in mild, biocompatible, metal-free conditions has been studied using Mannich-type additions to imines. These modifications, however, are subject to retro-Mannich type reactions. Therefore, there remains a need in the art for new methods for bioconjugation by modifying tyrosine residues in proteins.